*Target (protein/gene name): Alpha Carbonate Dehydratase *NCBI Gene # or RefSeq#:HPHPH27_0808 Change this - DR. B new gene iD: 899948 (9/13/13) *Protein ID (NP or XP #) or Wolbachia#: EJB55340 - Get the WP # for this here. - Dr. B090213
NP #: NC_000915 http://www.ncbi.nlm.nih.gov/gene/899948
(use Summer13E Plate for Oligos instead of ordering new ones - Dr. B 090313)) - Used Summer13E Plate for Oligos - 091313
*Organism (including strain):Helicobacter pylori Etiologic Risk Group: Group I—Pathogens Newly Recognized in the Past Two Decades *Background/Disease Information: Helicobacter pylori is a spiral-shaped bacterium commonly found in the stomach where the shape of the bacteria allow them to penetrate the stomach's protective mucous lining, where substances produced weaken the lining and make the stomach more susceptible to damage from gastric acids. In fact, most people infected with the bacteria never have symptoms or problems such as ulcers. Only a small number of people with the infection develop stomach cancer. But the most common symptom of peptic ulcer disease is gnawing or burning abdominal pain, usually in the area just beneath the ribs. From research, it seems to be that the frequency of people infected may somehow be related to race. About 60% of Hispanics and about 54% of African Americans have detectable organisms as compared to about 20%-29% of Anglo Americans. It is also known that children are very commonly infected in developing countries.
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1KOP (for homology model)
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 89%
---- Max % Identities: 38%
---- % Positives: 50%
---- Chain used for homology:
Current Inhibitors: According to the article, acetazolamide and methazolamide were shown to inhibit the bacterial growth in vitro. http://www.ncbi.nlm.nih.gov/pubmed/18336307 Expression Information (has it been expressed in bacterial cells): Based off the article, The protein appears to be toxic to E. coli, so a modified form of the gene lacking a part was prepared that encodes a cleavable signal peptide. http://www.brenda-enzymes.org/literature/lit.php4?e=4.2.1.1&r=650349 Purification Method: In this article, the H. pylori enzyme was purified by sulfonamide affinity chromatography. http://www.brenda-enzymes.org/literature/lit.php4?e=4.2.1.1&r=650349 Image of protein (PyMol with features delineated and shown separately):
no structure for this protein. *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MKKTFLIALALTASLIGAENTKWDYKNKENGPHRWDKLHKDFEV CKSGKSQSPINIEHYYHTQDKADLQFKYAASKPKAVFFTHHTLKASFEPTNHINYRGH DYVLDNVHFHAPMEFLINNKTRPLSAHFVHKDAKGRLLVLAIGFEEGKENPNLDPILE SVQKKQNFKEVALDAFLPKSINYYHFNGSLTAPPCTEGVAWFVIEEPLEVSAKQLAEI KKRMKNSPNQRPVQPDYNTVIIKSSAETR *length of your protein in Amino Acids: 247 aa Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
28.3153 kDa Molar Extinction coefficient of your protein at 280 nm wavelength:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 30035 Abs 0.1% (=1 g/l) 1.061, assuming all pairs of Cys residues form cystines
Ext. coefficient 29910 Abs 0.1% (=1 g/l) 1.056, assuming all Cys residues are reduced TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). *CDS Gene Sequence (paste as text only): atgaaaaaaacttttttgatcgctttagcacttacggcttctcttataggcgctgaaaataccaaatgggattataaaaataaaga aaatggcccgcaccgttgggacaaattgcacaaagattttgaagtgtgcaaaagcggtaaaagccaatcgcccatcaacatt gagcattactaccacacgcaagataaagccgatttgcaattcaaatacgccgcttccaaacctaaagcggtctttttcacccat cacactttaaaggcttcgtttgagccgactaaccacatcaattatagagggcatgactatgtgctggataatgtgcatttccacg cccctatggagtttttaatcaataataaaaccaggcctttgagcgcgcatttcgtgcataaagacgctaaagggcgtttgttagt gttagcgattggttttgaagaagggaaagaaaaccccaaccttgatcctattttagaaagcgttcaaaagaaacaaaattttaa agaggtggctttagacgctttcttgcctaaaagcatcaattactaccattttaacggctctctcaccgctcctccttgcacagaggg ggtggcatggtttgtcatagaagagcctttggaagtctctgccaaacaattggctgaaatcaaaaaacgcatgaaaaattcgcctaa ccaacgccccgtccagcctgactacaacaccgtgatcattaaaagctcagctgagacccgctaa *GC% Content for gene: 42.3% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol:
Primer design results for pNIC-Bsa4 cloning (list sequences of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) added (9/13/13)- https://docs.google.com/file/d/0B_Gl3lMyhDsoYVNRdHI4ZjZnc2c/edit
Primer design results for 'tail' primers (this is just 2 sequences): - added (9/13/13) Forward Primer: 5’ TACTTCCAATCCATGAAAAAAACCTTCCTG 3’ 30 bp GC Content=36.7% 0mM Mg2+ Tm=58.1°C 1.5mM Mg2+ Tm=65.8ºC 2mM Mg2+ Tm=66.4ºC 4mM Mg2+ Tm=67.5ºC 6mM Mg2+ Tm=68.1ºC
Enter the Template PDB ID: 1KOPB Enter the Save the Identities 37% and Positives 50% Enter the Molprobity scores for both the Template and the Model: Template: Clash score = 6.26 Molprobity score = 1.64 Model: Clash score= 16.3 Molprobity score = 2.90
10/31/13 Kelly K. Enter the Template PDB ID: 4G7A Enter the Save the Identities 39% and Positives 55% Ener the Molprobity scores for both the Template and the Model: Template: Clash score = 6.26 Molprobity score = 1.50 Model: Clash score = 29.99 Molprobity score = 3.23
*NCBI Gene # or RefSeq#: HPHPH27_0808 Change this - DR. B new gene iD: 899948 (9/13/13)
*Protein ID (NP or XP #) or Wolbachia#: EJB55340 - Get the WP # for this here. - Dr. B090213
NP #: NC_000915 http://www.ncbi.nlm.nih.gov/gene/899948
(use Summer13E Plate for Oligos instead of ordering new ones - Dr. B 090313))
- Used Summer13E Plate for Oligos - 091313
*Organism (including strain): Helicobacter pylori
Etiologic Risk Group: Group I—Pathogens Newly Recognized in the Past Two Decades
*Background/Disease Information:
Helicobacter pylori is a spiral-shaped bacterium commonly found in the stomach where the shape of the bacteria allow them to penetrate the stomach's protective mucous lining, where substances produced weaken the lining and make the stomach more susceptible to damage from gastric acids.
In fact, most people infected with the bacteria never have symptoms or problems such as ulcers. Only a small number of people with the infection develop stomach cancer. But the most common symptom of peptic ulcer disease is gnawing or burning abdominal pain, usually in the area just beneath the ribs.
From research, it seems to be that the frequency of people infected may somehow be related to race. About 60% of Hispanics and about 54% of African Americans have detectable organisms as compared to about 20%-29% of Anglo Americans. It is also known that children are very commonly infected in developing countries.
Essentiality of this protein: According to the article found, this protein is essential because carbonic anhydrases contributes to acid acclimation.
http://www.brenda-enzymes.org/literature/lit.php4?e=4.2.1.1&r=665394
Complex of proteins?:
*EC#: 4.2.1.1
Link to BRENDA EC# page: http://www.brenda-enzymes.org/php/result_flat.php4?ecno=4.2.1.1
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents): All information contained in this link.
http://www.sigmaaldrich.com/technical-documents/protocols/biology/enzymatic-assay-of-carbonic-anhydrase.html
-- link to Sigma (or other company) page for assay or assay reagents (substrates)
-- link (or citation) to paper that contains assay information
-- List cost and quantity of substrate reagents and supplier
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 1KOP (for homology model)
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 89%
---- Max % Identities: 38%
---- % Positives: 50%
---- Chain used for homology:
Current Inhibitors:
According to the article, acetazolamide and methazolamide were shown to inhibit the bacterial growth in vitro.
http://www.ncbi.nlm.nih.gov/pubmed/18336307
Expression Information (has it been expressed in bacterial cells): Based off the article,
The protein appears to be toxic to E. coli, so a modified form of the gene lacking a part was prepared that encodes a cleavable signal peptide.
http://www.brenda-enzymes.org/literature/lit.php4?e=4.2.1.1&r=650349
Purification Method:
In this article, the H. pylori enzyme was purified by sulfonamide affinity chromatography.
http://www.brenda-enzymes.org/literature/lit.php4?e=4.2.1.1&r=650349
Image of protein (PyMol with features delineated and shown separately):
no structure for this protein.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MKKTFLIALALTASLIGAENTKWDYKNKENGPHRWDKLHKDFEV
CKSGKSQSPINIEHYYHTQDKADLQFKYAASKPKAVFFTHHTLKASFEPTNHINYRGH
DYVLDNVHFHAPMEFLINNKTRPLSAHFVHKDAKGRLLVLAIGFEEGKENPNLDPILE
SVQKKQNFKEVALDAFLPKSINYYHFNGSLTAPPCTEGVAWFVIEEPLEVSAKQLAEI
KKRMKNSPNQRPVQPDYNTVIIKSSAETR
*length of your protein in Amino Acids: 247 aa
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website
28.3153 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 30035 Abs 0.1% (=1 g/l) 1.061, assuming all pairs of Cys residues form cystines
Ext. coefficient 29910 Abs 0.1% (=1 g/l) 1.056, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html).
*CDS Gene Sequence (paste as text only):
atgaaaaaaacttttttgatcgctttagcacttacggcttctcttataggcgctgaaaataccaaatgggattataaaaataaaga
aaatggcccgcaccgttgggacaaattgcacaaagattttgaagtgtgcaaaagcggtaaaagccaatcgcccatcaacatt
gagcattactaccacacgcaagataaagccgatttgcaattcaaatacgccgcttccaaacctaaagcggtctttttcacccat
cacactttaaaggcttcgtttgagccgactaaccacatcaattatagagggcatgactatgtgctggataatgtgcatttccacg
cccctatggagtttttaatcaataataaaaccaggcctttgagcgcgcatttcgtgcataaagacgctaaagggcgtttgttagt
gttagcgattggttttgaagaagggaaagaaaaccccaaccttgatcctattttagaaagcgttcaaaagaaacaaaattttaa
agaggtggctttagacgctttcttgcctaaaagcatcaattactaccattttaacggctctctcaccgctcctccttgcacagaggg
ggtggcatggtttgtcatagaagagcctttggaagtctctgccaaacaattggctgaaatcaaaaaacgcatgaaaaattcgcctaa
ccaacgccccgtccagcctgactacaacaccgtgatcattaaaagctcagctgagacccgctaa
*GC% Content for gene: 42.3%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol:
SEQUENCE 1: PROTEIN LENGTH = 248
----------------------------------------------------------------
1 MKKTFLIALVLATSLIGAENAKWDYKNKENGPHRWDKLHKDFEVCKSGKSQSPINIEHYY
61 HTQDKADLQFKYAASKPKAVFFTHHTLKASFEPTNHINYRGHDYVLDNVHFHAPMEFLIN
121 NKTRPLSAHFVHKDAKGRLLVLAIGFEEGKENPNLDPILEGIQKKQNFKEVALDAFLPKS
181 INYYHFNGSLTAPPCTEGVAWFVIEEPLEVSAKQLAEIKKRMKNSPNQRPVQPDYNTVII
241 KSSAKTRX
1 ATGAAAAAAACCTTCCTGATCGCGCTGGTGCTGGCGACCTCTCTGATCGGTGCGGAGAAC
61 GCCAAATGGGACTACAAAAACAAAGAGAATGGTCCTCACCGTTGGGACAAACTGCACAAA
121 GACTTTGAAGTATGCAAGTCTGGTAAATCTCAGTCTCCGATCAACATCGAACACTACTAT
181 CACACCCAGGACAAAGCGGACCTGCAGTTCAAATACGCGGCGTCTAAACCGAAAGCGGTT
241 TTCTTCACCCACCACACCCTGAAAGCGTCTTTCGAACCGACCAACCACATCAACTACCGT
301 GGTCACGACTACGTTCTGGACAACGTTCACTTCCACGCGCCGATGGAATTTCTGATTAAC
361 AACAAAACCCGTCCGCTGTCTGCGCACTTCGTTCACAAGGACGCGAAAGGTCGTCTGCTG
421 GTTCTGGCAATCGGTTTCGAAGAAGGTAAAGAAAATCCTAACCTGGACCCGATCCTGGAA
481 GGTATCCAAAAGAAACAGAACTTTAAGGAAGTTGCGCTGGATGCGTTTCTCCCGAAATCT
541 ATCAATTACTACCACTTCAACGGCTCTCTCACGGCCCCTCCTTGCACCGAAGGTGTTGCG
601 TGGTTTGTTATCGAAGAGCCACTGGAGGTCTCTGCGAAACAGCTGGCGGAAATCAAAAAA
661 CGTATGAAGAACTCTCCGAACCAACGCCCGGTGCAGCCGGATTATAACACCGTTATCATC
721 AAATCTTCTGCAAAGACCCGTTAA
18 oligonucleotides need to be synthesized
----------------------------------------------------------------
1 ATGAAAAAAACCTTCCTGATCGCGCTGGTGCTGGCGACCTCTCTGATCGGTGCGGA 56
2 TGAGGACCATTCTCTTTGTTTTTGTAGTCCCATTTGGCGTTCTCCGCACCGATCAGAGAG 60
3 AAAAACAAAGAGAATGGTCCTCACCGTTGGGACAAACTGCACAAAGACTTTGAAGTATGC 60
4 GTTCGATGTTGATCGGAGACTGAGATTTACCAGACTTGCATACTTCAAAGTCTTTGTGCA 60
5 AGTCTCCGATCAACATCGAACACTACTATCACACCCAGGACAAAGCGGACCTGCAGTTCA 60
6 TGGTGGGTGAAGAAAACCGCTTTCGGTTTAGACGCCGCGTATTTGAACTGCAGGTCCGCT 60
7 CGGTTTTCTTCACCCACCACACCCTGAAAGCGTCTTTCGAACCGACCAACCACATCAACT 60
8 GTGGAAGTGAACGTTGTCCAGAACGTAGTCGTGACCACGGTAGTTGATGTGGTTGGTCGG 60
9 TGGACAACGTTCACTTCCACGCGCCGATGGAATTTCTGATTAACAACAAAACCCGTCCGC 60
10 GCAGACGACCTTTCGCGTCCTTGTGAACGAAGTGCGCAGACAGCGGACGGGTTTTGTTGT 60
11 CGCGAAAGGTCGTCTGCTGGTTCTGGCAATCGGTTTCGAAGAAGGTAAAGAAAATCCTAA 60
12 GTTTCTTTTGGATACCTTCCAGGATCGGGTCCAGGTTAGGATTTTCTTTACCTTCTTCGA 60
13 CCTGGAAGGTATCCAAAAGAAACAGAACTTTAAGGAAGTTGCGCTGGATGCGTTTCTCCC 60
14 GCCGTGAGAGAGCCGTTGAAGTGGTAGTAATTGATAGATTTCGGGAGAAACGCATCCAGC 60
15 AACGGCTCTCTCACGGCCCCTCCTTGCACCGAAGGTGTTGCGTGGTTTGTTATCGAAGAG 60
16 TTTGATTTCCGCCAGCTGTTTCGCAGAGACCTCCAGTGGCTCTTCGATAACAAACCACGC 60
17 ACAGCTGGCGGAAATCAAAAAACGTATGAAGAACTCTCCGAACCAACGCCCGGTGCAGCC 60
18 TTAACGGGTCTTTGCAGAAGATTTGATGATAACGGTGTTATAATCCGGCTGCACCGGGCG 60
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list sequences of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
added (9/13/13)- https://docs.google.com/file/d/0B_Gl3lMyhDsoYVNRdHI4ZjZnc2c/edit
Primer design results for 'tail' primers (this is just 2 sequences): - added (9/13/13)
Forward Primer:
5’ TACTTCCAATCCATGAAAAAAACCTTCCTG 3’
30 bp
GC Content=36.7%
0mM Mg2+ Tm=58.1°C
1.5mM Mg2+ Tm=65.8ºC
2mM Mg2+ Tm=66.4ºC
4mM Mg2+ Tm=67.5ºC
6mM Mg2+ Tm=68.1ºC
Reverse Primer:
5’ TATCCACCTTTACTGTTAACGGGTCTTTGCAGA 3’
33 bp
GC Content=42.4%
0mM Mg2+ Tm=62.4°C
1.5mM Mg2+ Tm=69.8ºC
2mM Mg2+ Tm=70.3ºC
4mM Mg2+ Tm=71.3ºC
6mM Mg2+ Tm=71.8ºC
(9/14/13)
Forward Primer:
TACTTCCAATCCATGAAAAAAACTTTCCTGATCGCGC
37 bp
GC Content: %
2mM Mg2+ Tm: 71.5 Celsius
Reverse Compliment (Downstream):
TATCCACCTTTACTGACGGGTTTTCGCAGAAGA
33 bp
GC Content: %
2 mM Mg2+ Tm: 71.5 Celsius
References:
http://www.medicinenet.com/helicobacter_pylori/page2.htm#what_does_h_pylori_cause_in_humans
http://www.webmd.com/digestive-disorders/h-pylori-helicobacter-pylori
10/25/13 QasimA.
Enter the Template PDB ID: 1KOPB
Enter the Save the Identities 37% and Positives 50%
Enter the Molprobity scores for both the Template and the Model:
Template: Clash score = 6.26 Molprobity score = 1.64
Model: Clash score= 16.3 Molprobity score = 2.90
10/31/13 Kelly K.
Enter the Template PDB ID: 4G7A
Enter the Save the Identities 39% and Positives 55%
Ener the Molprobity scores for both the Template and the Model:
Template: Clash score = 6.26 Molprobity score = 1.50
Model: Clash score = 29.99 Molprobity score = 3.23